10  Pseudobulk

Run reintegration after cleaning

Published

August 28, 2025

10.1 REINTEGRATION

The integrated scRNAseq dataset of all brain cell populations was subsetted to retain those cells no subsetted out by the “expert committee”.

10.2 PCA

PC details

  1. PC1 captures a strong myelination axis, with high loadings for oligodendrocyte and myelin-related genes (Plp1, Mbp, Mag, Mal, Car2, Cryab) and opposite contributions from astrocytic (Ptgds, Enpp2, Edil3) and microglial (Hexb, C1qa, Ctss, Cst3) markers.

  2. PC2 reflects a neuron–astrocyte interaction gradient, driven by the astrocytic ECM regulator Sparcl1 on one pole and the neuronal signaling gene Plcb1 on the other.

  3. PC3 highlights synaptic connectivity and neurotransmission, with neuronal adhesion and plasticity genes (Nrxn1, Ntm, Lsamp, Ctnnd2, Adgrb3, Ntrk2) and the inhibitory receptor Gabrb1 loading strongly, balanced by astrocytic transporters (Slc1a2, Slc4a4) and Ptprz1. Together, these axes describe complementary layers of variance corresponding to myelination, glial–neuronal signaling, and synaptic specialization.

10.3 UMAP after reintegration

Adu IgG p.overall
N=3 N=3
Astrocytes 5.90 [3.90;8.90] 7.70 [5.95;8.05] 0.827
BAM 0.90 [0.85;0.90] 0.40 [0.40;0.65] 0.239
Choroid 2.30 [1.35;2.50] 1.60 [0.90;2.70] 0.827
Endothelial 7.20 [7.20;7.45] 7.60 [7.30;9.65] 0.825
Ependymal 0.30 [0.25;0.45] 0.60 [0.45;0.65] 0.261
Microglia 58.9 [56.3;62.3] 59.1 [57.0;59.5] 0.827
NPC 0.70 [0.55;0.85] 1.60 [1.45;1.70] 0.050
Neurons 1.10 [0.85;1.70] 1.10 [0.85;1.85] 0.822
OPC 0.30 [0.25;0.30] 0.60 [0.40;0.75] 0.369
Oligodendrocytes 17.2 [15.1;21.5] 17.2 [16.9;17.5] 1.000
T-cell 1.80 [1.65;1.85] 1.70 [1.65;1.90] 0.827
VMC 0.90 [0.70;1.00] 1.10 [1.05;1.10] 0.246

10.4 Pseudobulk

10.4.1 Microglia

p_val avg_log2FC pct.1 pct.2 p_val_adj
H2ac20 0.0e+00 -0.1750223 1 1 0.0000036
mt-Nd4l 0.0e+00 -0.4711669 1 1 0.0001628
Tnfaip2 0.0e+00 -0.4571713 1 1 0.0001815
Gm13849 0.0e+00 -0.0263631 1 1 0.0004293
Prdm1 1.0e-07 -0.1948147 1 1 0.0020465
C430049B03Rik 2.0e-07 -0.1446463 1 1 0.0032855
Man1a 2.0e-07 -0.2427612 1 1 0.0042481
Ccl7 3.0e-07 0.0704861 1 1 0.0071494
Il1a 4.0e-07 -0.5749078 1 1 0.0077532
Cxcl1 6.0e-07 -0.2383173 1 1 0.0136628
Dusp2 1.1e-06 -0.3441218 1 1 0.0242598
Rpl35 1.4e-06 -0.3722164 1 1 0.0304758
Mir155hg 1.5e-06 -0.2601184 1 1 0.0321594
mt-Atp8 1.9e-06 -0.2756153 1 1 0.0409223
Slc38a2 1.9e-06 -0.1951182 1 1 0.0417004

Following treatment, microglia from the Alzheimer’s disease mouse model exhibited a broad transcriptional shift consistent with reduced pro-inflammatory activation and metabolic demand. Several mitochondrial genes (mt-Nd4l, mt-Atp8) and the amino acid transporter Slc38a2 were downregulated, suggesting diminished oxidative phosphorylation and nutrient uptake that normally support highly activated microglial states. Key inflammatory mediators, including Il1a, Cxcl1, Tnfaip2, and the microRNA host gene Mir155hg, also decreased, indicating attenuation of cytokine release, chemotactic signaling, NF-κB pathway activity, and miR-155–driven immune activation. Concomitant reductions in Prdm1 and regulatory non-coding transcripts (Gm13849, C430049B03Rik), together with decreased Rpl35 and Man1a, point to lower transcriptional activity, protein synthesis, and secretory function, while the decline in H2ac20 implies chromatin remodeling toward a less plastic state. Notably, this general dampening of inflammatory pathways was accompanied by increased expression of Ccl7, a chemokine involved in monocyte recruitment. This suggests that while most pro-inflammatory signals were suppressed, the treatment selectively maintained or enhanced pathways favoring controlled immune cell trafficking, potentially reflecting a rebalancing of microglial responses rather than their complete silencing. Collectively, these findings support the notion that the treatment shifts microglia away from a broadly activated phenotype toward a more homeostatic yet immunologically competent state, which may contribute to mitigation of chronic neuroinflammation in Alzheimer’s disease.

10.4.2 Astrocytes

p_val avg_log2FC pct.1 pct.2 p_val_adj
Wdr17 1.0e-07 -0.6656742 1 1 0.0011904
Lpar1 1.0e-07 0.5192552 1 1 0.0012595
Dcc 1.0e-07 -0.6273549 1 1 0.0030505
Wif1 7.0e-07 0.1848369 1 1 0.0159661
Sorcs1 1.1e-06 0.3837130 1 1 0.0234440
Tmsb4x 1.8e-06 0.8196337 1 1 0.0383954
Tenm2 2.2e-06 0.3594390 1 1 0.0476388

10.4.3 BAM

p_val avg_log2FC pct.1 pct.2 p_val_adj
Cxcl13 0e+00 1.9569000 1 0.333 0.0002040
Wfdc17 2e-06 -0.8501451 1 1.000 0.0435564

10.4.4 Choroid

p_val avg_log2FC pct.1 pct.2 p_val_adj
Rgs2 0 0.9370808 1 1 0.00068

10.4.5 Endothelial

p_val avg_log2FC pct.1 pct.2 p_val_adj
Gria1 1e-07 0.2876162 1 1 0.0014814
Slc38a2 5e-07 -0.3942290 1 1 0.0101714

10.4.6 Ependymal

p_val avg_log2FC pct.1 pct.2 p_val_adj
Mt3 1e-07 -0.581966 1 1 0.0014678

10.4.7 Neurons

p_val avg_log2FC pct.1 pct.2 p_val_adj
Cldn5 0.0e+00 0.8740228 1.000 1.000 0.0000026
Ly6c1 0.0e+00 0.8417682 1.000 1.000 0.0000039
Hapln1 0.0e+00 -1.0575334 0.667 1.000 0.0000206
Slco1a4 0.0e+00 0.9098885 1.000 0.667 0.0000294
Ptprb 0.0e+00 0.7883225 1.000 0.667 0.0003976
Vgf 1.1e-06 0.8479086 1.000 1.000 0.0243259
Ly6a 1.6e-06 0.7068322 1.000 0.667 0.0353316

10.4.8 NPC

p_val avg_log2FC pct.1 pct.2 p_val_adj
Ikzf1 0.0e+00 1.0571115 1 1 0.0000004
Runx1 0.0e+00 1.3537906 1 1 0.0000057
Creg1 0.0e+00 1.0016099 1 1 0.0000301
Gpr183 0.0e+00 1.1165005 1 1 0.0000624
Adamts1 0.0e+00 0.9092272 1 1 0.0001537
Inpp5d 0.0e+00 1.1274030 1 1 0.0001906
Zfp36 0.0e+00 1.0025476 1 1 0.0003610
Lmo2 0.0e+00 0.9614975 1 1 0.0003979
Lrmda 0.0e+00 0.9927620 1 1 0.0009119
Fth1 1.0e-07 0.7754402 1 1 0.0015168
Sat1 1.0e-07 1.1250506 1 1 0.0016771
Lpcat2 1.0e-07 0.9794508 1 1 0.0017612
Unc93b1 1.0e-07 1.1107730 1 1 0.0018391
Nfkbia 1.0e-07 0.8980613 1 1 0.0019855
Tmem119 1.0e-07 0.8763570 1 1 0.0020384
Ptprc 2.0e-07 0.9292133 1 1 0.0042320
Lgals9 3.0e-07 0.7196859 1 1 0.0058081
Adap2 3.0e-07 0.8139424 1 1 0.0071801
P2ry12 6.0e-07 1.0277527 1 1 0.0118773
Cd83 7.0e-07 0.9920499 1 1 0.0145304
Itgam 7.0e-07 1.0216380 1 1 0.0145724
Abca9 8.0e-07 0.8031022 1 1 0.0174620
Rnase4 8.0e-07 0.8590141 1 1 0.0179884
Ly6e 8.0e-07 0.8554275 1 1 0.0180234
H2-T23 1.6e-06 0.7770506 1 1 0.0350168
Selplg 1.6e-06 0.7707752 1 1 0.0351740
Lyn 1.8e-06 0.9601579 1 1 0.0397079
St3gal6 2.1e-06 0.8886279 1 1 0.0453242

10.4.9 Oligodendrocytes

p_val avg_log2FC pct.1 pct.2 p_val_adj
Ms4a6b 5.0e-07 0.0686229 1 1 0.0096832
C1qa 8.0e-07 0.4272693 1 1 0.0167962
Shoc2 9.0e-07 -0.2455762 1 1 0.0196189
Ctss 1.2e-06 0.4387682 1 1 0.0254450
mt-Atp8 1.3e-06 -0.3642960 1 1 0.0273779
mt-Nd4l 2.0e-06 -0.4666682 1 1 0.0426910

10.4.10 OPC

p_val avg_log2FC pct.1 pct.2 p_val_adj
Hpgd 5e-07 -1.446021 1 1 0.0109219

10.4.11 T-cell

p_val avg_log2FC pct.1 pct.2 p_val_adj
Dscam 4e-07 0.8264654 1 1 0.008977

10.4.12 VMC

p_val avg_log2FC pct.1 pct.2 p_val_adj
Rbp4 0 1.454712 1 0.667 0.0003589

10.4.13 Number of DEGs up/down-regululated by aducanumab